RNA Modifications as Biomarkers of Environmental Stress and Inflammation

描述(申请人提供)：拟议的研究有两个目的：加深我们对转录后tRNA修饰生物学的理解，并探索tRNA修饰作为导致癌症和其他疾病的慢性炎症病理生理学的生物标志物的用途。TRNA有几十种不同的碱基结构，但其中大多数修饰的确切功能尚不清楚。我们最近证明了tRNA中修饰尿苷(Mcm5U)的酶催化甲基化在DNA损伤后的密码子特异性翻译控制中发挥作用，我们有初步数据表明，mcm5U和其他tRNA修饰的水平在暴露于各种基因毒素后发生特异性变化。我们假设，酶催化的tRNA修饰的水平和模式可以用作毒物暴露和病理生理的生物标志物。我们的假设所固有的是一个新的提议，即酶催化的tRNA修饰是损伤信号通路的动态组成部分。我们将使用生物标记物研究来验证我们的假设，建立酶催化的tRNA修饰作为损伤信号通路的组成部分，并更好地了解催化修饰为人类tRNA的时间顺序和蛋白质系统。为了实现我们的目标，我们将在细胞暴露于烷化剂和活性氧氮物种(RON)之前和之后，对tRNA物种的谱、tRNA核苷酸碱基20修饰以及tRNA相关基因的表达进行定量评估。我们将评估超过23个人类tRNA修饰系统特异性基因、63个可通过阵列技术区分的tRNA物种以及超过25个碱基修饰的组合潜力，以检验我们的建议，即以tRNA为中心的指数是具有大动态范围的高度特异性的生物标记物。此外，以tRNA为中心的指数将被用来阐明将修饰结合到tRNA中的机制基础，并强调tRNA在细胞对有害刺激和病理生理条件(如炎症)的反应中的作用。目的1：建立定量测定核糖核酸碱基20修饰谱的方法。本研究的目的是建立定量测定tRNA中酶催化的碱基修饰的分析方法。我们将使用两种质谱学方法来量化RNA修饰：全球或非靶向调查和特定核苷的靶向量化。这里的策略是使用全局方法来定义在细胞应激期间经历数量变化的修饰的谱，并将修饰与特定的酶和途径相关联。如果在全球研究中确定了暴露“特征”，则可以通过有针对性的策略以更高的敏感度来量化个别修改。这些方法将用酵母tRNA进行开发和验证，然后应用于小鼠和人类细胞的tRNA。目的2：评估以tRNA为中心的测量方法作为暴露在细胞中的试剂特异性生物标志物的特征。这个基于细胞的目的是应用Aim#1的方法来评估tRNA作为细胞应激的生物标记物，并获得对人类tRNA修饰系统的机械洞察力。此外，这些研究将提供一个tRNA特异性改变的文库，可以转化为活体研究。首先，我们将对人类HEK293细胞暴露于DNA损伤剂甲烷磺酸甲酯(MMS)和过氧化氢(H_2O_2)后修饰核苷的变化进行详细的剂量-反应和时间过程研究。这些结果将指导随后对(1)与tRNA修饰酶相对应的转录本、(2)单个tRNA物种和(3)tRNA修饰的水平的测量，以确定以tRNA为中心的暴露特征。为了验证我们的假设，并作为AIM#3的SJL小鼠炎症研究的基准，我们将量化暴露于四种化学炎症介质(过氧化氢、一氧化氮、过氧亚硝酸盐和高氯)的培养SJL细胞株中的tRNA中心指数。在所有研究中，最终的定量数据将通过计算挖掘，以识别每种药物特有的生物标记物签名。此外，我们将使用我们的生物标记物数据来强调tRNA修饰在损伤信号中的作用，并提供对人类tRNA修饰系统的机械性见解。目的3：使用小鼠炎症模型来证明tRNA修饰在体内具有生物标志物的潜力。在AIM#2中应用于细胞的方法和由此产生的模型现在将被转化为SJL小鼠一氧化氮过度产生和炎症的模型。这里的目标是量化与tRNA修饰酶相对应的转录本，并表征暴露于SJL小鼠炎症产生的活性氧和氮物种的组织的tRNA修饰的谱。所得模式将与对照动物进行比较，以检验我们的假设，即以tRNA为中心的措施可以作为慢性炎症的高度敏感的体内生物标志物，并且以tRNA为中心的措施可以用于评估体内炎症部位发生的化学物质。

DESCRIPTION (provided by applicant): The proposed studies have two objectives: to further our understanding of the biology of posttranscriptional tRNA modifications and to explore the utility of the tRNA modifications as biomarkers of the pathophysiology of chronic inflammation leading to cancer and other diseases. There are dozens of different nucleobase structures in tRNA, yet the precise function of most of these modifications is unknown. We have recently demonstrated that enzyme-catalyzed methylation of a modified uridine (mcm5U) in tRNA plays a role in codon- specific translational control after DNA damage, and we have preliminary data demonstrating that the levels of mcm5U and other tRNA modifications specifically change after exposure to a variety of genotoxins. We hypothesize that the levels and patterns of enzyme-catalyzed tRNA modifications can be used as biomarkers of toxicant exposures and pathophysiologies. Inherent to our hypothesis is the novel proposal that enzyme-catalyzed tRNA modifications are dynamic components of damage signaling pathways. We will use biomarker studies to test our hypothesis, to establish enzyme-catalyzed tRNA modifications as components of damage signaling pathways, and to better understand the temporal order and protein systems that catalyze modifications into human tRNA. To accomplish our objectives, we will perform quantitative assessments of the spectrum of tRNA species, tRNA nucleobase 20 modifications, and the expression of tRNA-related genes, before and after exposure of cells to alkylating agents and reactive oxygen and nitrogen species (RONS). The combinatorial potential of more than 23 genes specific to human tRNA modification systems, 63 tRNA species distinguishable by array techniques, and more than 25 nucleobase modifications will be assessed to test our proposal that tRNA-centric indices are highly specific biomarkers with a large dynamic range. Further, tRNA-centric indices will be used to clarify the mechanistic basis by which modifications are incorporated into tRNA and to highlight the role of tRNA in the cellular response to noxious stimuli and pathophysiological conditions such as inflammation. Aim 1: Develop methods to quantify the spectrum of RNA nucleobase 20 modifications. The objective of this aim is to develop analytical methods for quantifying enzyme-catalyzed nucleobase modifications in tRNA. We will use two mass spectrometric approaches to quantify RNA modifications: global or untargeted surveys and targeted quantification of specific nucleosides. The strategy here is to use the global approach to define the spectrum of modifications undergoing quantitative changes during cell stress and to correlate modifications with specific enzymes and pathways. If an exposure "signature" is identified in global studies, the individual modifications can be quantified with greater sensitivity by a targeted strategy. The methods will be developed and validated with yeast tRNA and then applied to tRNA from mouse and human cells. Aim 2: Assessment of tRNA-centric measures as agent-specific biomarker signatures of exposure in cells. The objectives of this cell-based aim are to apply the methods of Aim #1 to assess tRNA as a biomarker of cellular stress and to gain mechanistic insight into human tRNA modification systems. Further, these studies will provide a library of tRNA-specific changes that can be translated into in vivo studies. First, we will perform detailed dose-response and time course studies for changes in modified nucleosides after exposure of human HEK293 cells to the DNA damaging agents, methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). The results will guide subsequent measurements of the levels of (1) transcripts corresponding to tRNA modification enzymes, (2) individual tRNA species, and (3) tRNA modifications for determination of tRNA- centric exposure signatures. To test our hypothesis and as a benchmark for the SJL mouse inflammation studies of Aim #3, we will quantify tRNA-centric indices in cultured SJL cell lines exposed to four chemical mediators of inflammation (H2O2, nitric oxide, peroxynitrite and HOCl). In all studies, the resulting quantitative data will be computationally mined to identify biomarker signatures specific to each agent. Further, we will use our biomarker data to highlight a role for tRNA modifications in damage signaling and to provide mechanistic insight into human tRNA modification systems. Aim 3: Use a mouse model of inflammation to demonstrate that tRNA modifications have in vivo biomarker potential. The methods applied to cells in Aim #2 and the resulting models will now be translated to the SJL mouse model of nitric oxide over-production and inflammation. The goal here is to quantify the transcripts corresponding to tRNA modification enzymes and to characterize the spectrum of tRNA modifications from tissues exposed to the reactive oxygen and nitrogen species arising from inflammation in the SJL mice. The resulting patterns will be compared to control animals to test our hypotheses that tRNA- centric measures can be used as highly sensitive in vivo biomarkers of chronic inflammation and that tRNA- centric measures can be used to assess the chemistry that occurs at sites of inflammation in vivo.